Lung disease continues to be the major cause of morbidity and mortality in cystic fibrosis (CF). Gene transfer offers the potential to express the cystic fibrosis transmembrane conductance regulator (CFTR) in the lungs of people with cystic fibrosis (CF) and thereby prevent or treat lung disease. But, we lack answers to many questions that are crucial for developing gene transfer. A major impediment to progress has been lack of animal models with disease that mimics human CF. To overcome this roadblock, we developed a porcine model of CF that spontaneously develops lung disease. We discovered that loss of CFTR impairs the activity of antimicrobials in airway surface liquid (ASL) and hinders mucociliary transport (MCT). In both cases, loss of CFTR-mediated bicarbonate secretion and an abnormally acidic pH are key factors. We also developed novel assays of these host defense mechanisms for both in vitro and in vivo experiments. We will use this novel model and assays to answer questions that are key for advancing CF treatments. First, How does CFTR expression level and the fraction of cells expressing CFTR affect airway epithelial ion transport and host defense processes? Answering this question will for the first time provide key data about relationships between specific CFTR functions and host defense. Second, will CFTR expression correct host defense defects and prevent and/or treat CF lung disease in vivo? We will develop CF pigs with inducible expression of CFTR in epithelia and test the hypotheses a) that expressing CFTR in airways rescues host defense processes, b) that CFTR expression beginning shortly after birth prevents airway infection, inflammation and remodeling, c) that expressing less than wild-type levels of CFTR in airway epithelia is sufficient to prevent lung disease, and d) that CFTR expression after disease onset treats or reverses airway disease. Third, will expressing CFTR in ciliated cells rescue host defense defects and prevent clinical CF lung disease? If ciliated cell expression prevents lung disease, it will mark ciliated cells as a key target for gene transfer. If not, the data will guide studies that identify key therapeutic targets. Results from this work and synergism with Projects 1 & 3 will inform strategies for gene transfer and other therapies.